This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Our overall program correlates X-ray diffraction (XRD, of glycerinated native fibers) and thin-section EM tomography (fibers quick-frozen &freeze-substituted) to characterize structure, arrangement and dynamic choreography of myosin crossbridges (muscle[unreadable]s motor molecules) in a waterbug insect flight muscle (IFM) of unexcelled crystalline regularity. X- ray patterns are correlated with parallel EM of fibers quick-frozen during the same equilibrium state, perturbation or transition, to image directly the shapes and patterns of crossbridges, clearer in this muscle than any other. Recent progress (attached) has poised us to suggest the unknown structural trigger for IFM[unreadable]s famous stretch-activation (SA) process, which may also throw light on the similar process in mammalian heart muscle. A published gallery (PNAS, 2008) of XRD patterns from 3 major IFM states, RLX, ACT &RIG, shows the same ACT structure reached by 2 distinct RLX[unreadable]ACT pathways, Ca2+ -activation (at pCa 4.5) and SA (at pCa 5.7). Striking new XRD "movies" of the cycling work-loop form of SA (Biophysics Abst, 2009), driven by sinusoidal 2%/2Hz length changes, show cycling intensity changes in multiple reflections that signal well-known major actions by crossbridges and tropomyosin. An alternate single-shot form of SA, triggered by a stretch-and-hold length-step, is now supporting the model suggested by the work-loop movies, indicating that tropomyosin moves into ON position before crossbridges begin to attach and generate force.